Degradable heterobifunctional poly(ethylene glycol) acrylates and gels and  conjugates derived therefrom

ABSTRACT

A heterobifunctional poly(ethylene glycol) is provided having a hydrolytically degradable linkage, a first terminus comprising an acrylate group, and a second terminus comprising a target such as a protein or pharmaceutical agent or a reactive moiety capable of coupling to a target. Hydrogels can be prepared. The hydrogels can be used as a carrier for a protein or a pharmaceutical agent that can be readily released in a controlled fashion.

CROSS REFERENCE TO RELATED APPLICATION

This is a continuation application of application Ser. No. 12/860,525,filed Aug. 20, 2010, now abandoned, which is a continuation ofapplication Ser. No. 11/745,235, filed May 7, 2007, now abandoned, whichis a continuation application of application Ser. No. 10/684,692, filedOct. 14, 2003, now U.S. Pat. No. 7,214,388, which is a continuationapplication of application Ser. No. 09/824,395, filed Apr. 2, 2001, nowabandoned, which is a divisional application of application Ser. No.09/226,341, filed Jan. 6, 1999, now U.S. Pat. No. 6,362,276, which isrelated to commonly owned Provisional Application Ser. No. 60/070,680,filed Jan. 7, 1998, and claims the benefit of its filing date under 35USC Section 119(e), all of the applications are incorporated byreference in their entireties.

FIELD OF THE INVENTION

This invention relates to heterobifunctional poly(alkylene oxides)having degradable linkages and to conjugates derived therefrom.

BACKGROUND OF THE INVENTION

In its most common form, the poly(alkylene oxide) poly(ethylene glycol)(PEG) is a linear polymer terminated at each end with hydroxyl groups:HO—CH₂CH₂O—(CH₂CH₂O)_(n)—CH₂CH₂—OHThis polymer can be represented in a brief form as HO-PEG-OH where it isunderstood that -PEG- represents the following structural unit:—CH₂CH₂O—(CH₂CH₂O)_(n)—CH₂CH₂—where n typically ranges from approximately 10 to 2000.

PEG is of great utility in a variety of biotechnical and pharmaceuticalapplications, particularly for drug delivery and modification of drugsurfaces to promote nonfouling characteristics.

PEG is not toxic, does not tend to promote an immune response, and issoluble in water and in many organic solvents. The PEG polymer can becovalently attached to insoluble molecules to make the resultingPEG-molecule conjugate soluble. For example, Greenwald, Pendri andBolikal in J. Org. Chem., 60, 331-336 (1995) recite that thewater-insoluble drug taxol, when coupled to PEG, becomes water soluble.Davis et al. in U.S. Pat. No. 4,179,337 recite that proteins coupled toPEG have an enhanced blood circulation lifetime because of a reducedrate of kidney clearance and reduced immunogenicity. The lack oftoxicity of the polymer and its rate of clearance from the body areimportant considerations in pharmaceutical applications. Pharmaceuticalapplications and many leading references are described in the book byHarris (J. M. Harris, Ed., “Biomedical and Biotechnical Applications ofPolyethylene Glycol Chemistry, Plenum, New York, 1992).

PEG is commonly used as methoxy-PEG-OH, or mPEG in brief, in which oneterminus is the relatively inert methoxy group, while the other terminusis a hydroxyl group that is subject to ready chemical modificationCH₃O—(CH₂CH₂O)_(n)—CH₂CH₂—OH mPEG

PEG is also commonly used in branched forms that can be prepared byaddition of ethylene oxide to various polyols, including glycerol,pentaerythritol and sorbitol. For example, the four-armed branched PEGprepared from pentaerythritol is shown below:C(CH₂OH)_(n) +nC₂H₄O→C[CH₂O—(CH₂CH₂O)_(n)—CH₂CH₂—OH]₄

The branched PEGs can be represented in a general form as R(-PEG-OH)_(n)in which R represents the central core molecule, which can include,e.g., glycerol or pentaerythritol, and n represents the number of arms.

Often it is necessary to use an “activated derivative” of PEG to couplePEG to a molecule. The hydroxyl group located at the PEG terminus, orother group subject to ready chemical modification, is activated bymodifying or replacing the group with a functional group suitable forreacting with a group on another molecule, including, e.g., proteins,surfaces, enzymes, and others. For example, the succinimidyl “activeester” of carboxymethylated PEG forms covalent bonds with amino groupson proteins as described by K. Iwasaki and Y. Iwashita in U.S. Pat. No.4,670,417. The synthesis described in U.S. Pat. No. 4,670,417 isillustrated below with the active ester reacting with amino groups of aprotein in which the succinimidyl group is represented as NHS and theprotein is represented as PRO-NH₂:PEG-O—CH₂—CO₂—NHS+PRO-NH₂→PEG-O—CH₂—CO₂—NH-PROSuccinimidyl “active esters”, such as PEG-OCH₂—CO₂—NHS, are commonlyused forms of activated carboxylic acid PEGs, and they are prepared byreacting carboxylic acid PEGs with N-hydroxysuccinimide.

PEG hydrogels, which are water-swollen gels, have been used for woundcovering and drug delivery. PEG hydrogels are prepared by incorporatingthe soluble, hydrophilic polymer into a chemically crosslinked networkor matrix so that addition of water produces an insoluble, swollen gel.Substances useful as drugs typically are not covalently attached to thePEG hydrogel for in vivo delivery. Instead, the substances are trappedwithin the crosslinked matrix and pass through the interstices in thematrix. The insoluble matrix can remain in the body indefinitely, andcontrol of the release of the drug typically can be somewhat imprecise.

One approach to preparation of these hydrogels is described by Embreyand Grant in U.S. Pat. No. 4,894,238. The ends of the linear polymer areconnected by various strong, nondegradable chemical linkages. Forexample, linear PEG is incorporated into a crosslinked network byreacting with a triol and a diisocyanate to form hydrolytically stableurethane linkages that are nondegradable in water.

A related approach for preparation of PEG hydrogels has been describedby Gayet and Fortier in J. Controlled Release, 38, 177-184 (1996) inwhich linear PEG was activated as the p-nitrophenylcarbonate andcrosslinked by reaction with a protein, bovine serum albumin. Thelinkages formed are hydrolytically stable urethane groups and thehydrogels are nondegradable in water.

In another approach, described by N. S. Chu in U.S. Pat. No. 3,963,805,nondegradable PEG networks have been prepared by random entanglement ofPEG chains with other polymers formed by use of free radical initiatorsmixed with multifunctional monomers. P. A. King described nondegradablePEG hydrogels in U.S. Pat. No. 3,149,006 that have been prepared byradiation-induced crosslinking of high molecular weight PEG.

Nagaoka et al. described in U.S. Pat. No. 4,424,311 preparing PEGhydrogels by copolymerization of PEG methacrylate with other comonomerssuch as methyl methacrylate. Vinyl polymerization produces apolyethylene backbone with PEG attached. The methyl methacrylatecomonomer is added to give the gel additional physical strength.

Sawhney et al. described, in Macromolecules, 26, 581 (1993) and U.S.Pat. No. 5,626,863, the preparation of block copolymers of polyglycolideor polylactide and PEG that are terminated with acrylate groups:CH₂═CH—CO—(O—CHR—CO)_(n)—O-PEG-O—(CO—CHR—O)_(n)—OC—CH═CH₂where R is CH₃— or H.

In the above formula, the glycolide blocks are the —OCH₂—CO— units;addition of a methyl group to the methylene group gives rise to alactide block; n can be multiples of 2. Vinyl polymerization of theacrylate groups produces an insoluble, crosslinked gel with apolyethylene backbone. The polylactide or polyglycolide segments of thepolymer backbone shown above, which are ester groups, are susceptible toslow hydrolytic breakdown, with the result that the crosslinked gelundergoes slow degradation and dissolution. While this approach providesfor degradable hydrogels, the structure provides no possibility ofcovalently attaching proteins or other drugs to the hydrogel forcontrolled release. Applications of these hydrogels in drug delivery arethus restricted to release of proteins or other drugs physicallyentrapped within the hydrogel, thus reducing the potential foradvantageous manipulation of release kinetics.

Hubbell, Pathak, Sawhney, Desai, and Hill (U.S. Pat. No. 5,410,016,1995) polymerized:Protein-NH-PEG-O₂C—CH═CH2with long wavelength uv radiation to obtain a PEG acrylate polymer witha protein linked to it. The link between the PEG and the protein was notdegradable, so the protein could only be hydrolytically released withPEG attached. Since the acrylate polymer is not hydrolyticallydegradable, the release of the PEG protein derivative is notcontrollable.

Yang, Mesiano, Venkatasubramanian, Gross, Harris and Russell in J. Am.Chem. Soc. 117, 4843-4850, (1995) described heterobifunctionalpoly(ethylene glycols) having an acrylate group on one terminus and anactivated carboxylic acid on the second terminus. They demonstrated theattachment of this PEG derivative to a protein and incorporation of theresulting PEG protein derivative into an acrylate polymer. However, thePEG backbone there is not degradable and the protein was thus, ineffect, permanently bound to the acrylate polymer.

SUMMARY OF THE INVENTION

This invention provides heterobifunctional acrylates of poly(alkyleneoxides), especially poly(ethylene glycol) (PEG) acrylates havinglinkages that are hydrolytically degradable and conjugates prepared fromthese acrylates having target materials such as proteins covalentlylinked thereto. Hydrogels can also be prepared from these acrylates. Thetarget materials can be released from the hydrogels through controllablehydrolytic degradation of the hydrogels.

In one embodiment of the invention, heterobifunctional PEG is providedwhich is represented by the formula:CH₂═CZ—CO₂-PEG-W-Qwhere Z is an alkyl group or hydrogen atom, W is a hydrolyticallyunstable linkage comprising a hydrolyzable covalent bond, and Q is areactive moiety capable of reacting with a target to form a covalentlinkage thus linking the PEG polymer to the target.

In another embodiment, this invention also provides a heterobifunctionalPEG with a hydrolyzable linkage W in the PEG backbone and having anacrylate group at one terminus and a reactive moiety Q at the otherterminus. The heterobifunctional PEG is represented by the formula of:CH₂═CZ—CO₂-PEG-W-PEG-Qwhere Z is an alkyl group or hydrogen atom, W is a hydrolyticallyunstable linkage comprising a hydrolyzable bond, and Q is a reactivemoiety capable of reacting with a moiety on a target such as protein ora drug.

The present invention also encompasses a conjugate having a formula of:(CH₂═CZ—CO₂-PEG-W-L)_(x)-Twhere Z and W are as described above, T is a target, e.g., a protein ora drug, L is a covalent linkage formed in the reaction between Q and areactive moiety of T, and x is a number from 1 to 10.

In yet another embodiment of the invention, a conjugate ofheterobifunctional PEG and a target is provided having the formula(CH₂═CZ—CO₂-PEG-W-PEG-L)_(x)-Twhere Z and W are as described above, T is a target, e.g., a protein ora drug, which is linked to the PEG polymer as a result of the reactionbetween the reactive moiety Q and a moiety on T, L is a covalent linkageformed in the reaction between Q and a reactive group of T, and x is anumber of from 1 to 10.

This invention further provides polymers formed by vinyl polymerizationof the aforementioned heterobifunctional PEG or conjugates thereof,represented by the formula: CH₂═CZ—CO₂-PEG-W-Q,(CH₂═CZ—CO₂-PEG-W-L)_(x)-T, CH₂═CZ—CO₂-PEG-W-PEG-Q, and(CH₂═CZ—CO₂-PEG-W-PEG-L)_(x)-T. The weak chemical linkages in the thusformed polymers provide for hydrolytic breakdown and concomitant releaseof bound target molecules. For example, polymerization of theabove-mentioned conjugate:(CH₂═CZ—CO₂-PEG-W-PEG-L)_(x)-Tyields a water-soluble acrylate polymer which upon hydrolyticdegradation liberates a smaller PEG fragment bearing a target such as aprotein or a drug.

In another embodiment of the invention, hydrogels are formed bycopolymerizing a heterobifunctional PEG conjugate of this invention witha PEG molecule having two or more acrylate groups (“PEG multiacrylate”).Exemplary examples of such PEG multiacrylate can be:CH₂═CHCO₂-PEG-O₂CCH═CH₂orCH₂═CHCO₂-PEG-O—CH₂CO₂CH(CH₃)CH₂CONH-PEGO₂CCH═CH₂The hydrogel of the present invention is a cross-linked network in whichprotein or other target molecules are covalently bound to a degradablematrix. Because of the hydrolytically unstable linkages W in thehydrogels, the target molecules such as drug or protein molecules can bereleased as a result of the breakdown of the unstable linkages.

In the heterobifunctional PEG, polymers, and hydrogels of the presentinvention, the hydrolytic breakdown of the hydrolytically unstablelinkages W can be controlled in part by varying W, in particular thenumber of methylene group proximate to the hydrolyzable bond in W.Specifically, as the number of methylene group increases, the hydrolysisrate of the hydrolyzable bond of W decreases.

Further, in the hydrogel of the present invention, the release rate ofthe target from the hydrogel can also be controlled by varying thenumber x in the above formula of the PEG conjugate, i.e., the number ofthe PEG acrylate linked to the target. The release rate of the targetfrom the hydrogel is decreased when the number of PEG acrylate linked tothe target is increased, and vice versa.

Thus, the present invention provides heterobifunctional PEG andhydrogels formed therefrom having target molecules covalently linked tothe hyrogels. In contrast to the PEG hydrogels known heretofore in theart, the target molecules can be released in a controlled fashion fromthe PEG hydrogels of the present invention. Further, since the releaserate of the target can be determined by both the number of the attachedPEG and the structure of the hydrolytically unstable linkage in theattached PEG, more precise control of the release kinetics is madepossible. Therefore, suitable drug carriers for drug delivery which meetdifferent drug release requirements can be made in accordance with thepresent invention.

The foregoing and other advantages and features of the invention, andthe manner in which the same are accomplished, will become more readilyapparent upon consideration of the following detailed description of theinvention taken in conjunction with the accompanying examples, whichillustrate preferred and exemplary embodiments.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a plot of the release profile of lucifer-yellow lysozyme froma PEG acrylate hydrogel.

DETAILED DESCRIPTION OF THE INVENTION

A heterobifunctional water soluble and hydrolytically degradable polymeris provided comprising a polymer backbone having a degradable linkage, afirst terminus comprising an acrylate group, and a second terminuscomprising a target or a functional group capable of coupling thepolymer to a target.

As used herein, the terms “group,” “moiety,” “site,” and “radical” areall somewhat synonymous and are used herein to refer to distinct,definable portions or units of a molecule or units that perform somefunction or activity or reactive with other molecules or portions ofmolecules.

The term “linkage” is used herein to refer to groups that normally areformed as the result of a chemical reaction and typically are covalentlinkages. Hydrolytically stable linkages means that the linkages arestable in water and do not react with water at useful pHs for anextended period of time, potentially indefinitely. Hydrolyticallyunstable linkages are those that react with water, typically causingdegradation of a hydrogel and release of substances trapped within thematrix. The linkage is said to be subject to hydrolysis and to behydrolyzable. The time it takes to degrade the crosslinked polymericstructure is referred to as the rate of hydrolysis and is usuallymeasured in terms of its half life. Thus, in the present invention, thetarget molecules typically are released at a predetermined rate orwithin a predetermined time.

“Heterobifunctional” refers to the first and second terminii on thepolymer, one of which is acrylate, and the other of which is the targetmolecule or or functional group capable of coupling the polymer to atarget.

A preferred embodiment of the heterobifunctional polymer is representedby the formula:CH₂═CZ—CO₂-POLY-W-POLY′-Q.Another preferred embodiment is represented by the formula:CH₂═CZCO₂-POLY-W-Q.

In the above formulas, Z can be H or an alkyl group. Preferably, thealkyl group has less than 20 carbon atoms, more preferably less than 10carbon atoms, and most preferably less than 3 carbon atoms.

Typically, the polymer backbone represented by POLY and POLY′ arepoly(alkylene oxide), including derivatives thereof. A suitablepoly(alkylene oxide) or derivative thereof can comprise a grouprepresented by the formula —(CH₂CHRO)_(n)—CH₂CHR— in which R is H or analkyl group, and n ranges from about 10 to about 4000. Preferably, R isH and the polymer backbone comprises a poly(ethylene glycol) group.Poly(ethylene glycol) is preferred because it is substantially non-toxicand non-immunogenic.

W is a hydrolytically unstable linkage that can break down in an aqueousenvironment by hydrolysis. Typically, the linkage W comprises ahydrolyzable covalent bond. Suitable examples of such hydrolyzablecovalent bonds include, but are not limited to, carboxylate esters,imines, phosphate esters, acetals, orthoesters, peptide bonds, andoligonucleotides.

These hydrolyzable bonds can be formed by reaction of pairs of reactivemoieties, for example, alcohol and carboxylic acid reacting to formcarboxylate esters, amine and aldehyde reacting to form imines,hydrazide and aldehyde reacting to form hydrazones, alcohol andphosphate reacting to form phosphate ester, aldehyde and alcoholreacting to from acetals, alcohols and formate reacting to formorthoesters, amino acid and amino acid reacting to form peptide bonds,nucleotide and nucleotide to form oligonucleotide bonds.

Typically the hydrolytically degradable linkage W further comprises aplurality of alkylene groups, preferably methylene groups, proximate tothe hydrolyzable bond. The rate of degradation of the hydrolyticallydegradable linkage W by hydrolysis is in part determined by the numberof the alkylene groups and the distance of these groups from thehydrolyzable bond.

In a preferred embodiment, W has the structure of:—O(CH₂)_(m)—CO₂R₁—CO₂—or—O(CH₂)_(m)—CO₂—,where m ranges from 1 to 10 and R₁ is selected from the group consistingof —CH₂—, —CH₂CH₂—, and —CH(CH₃)CH₂—. In these two examples, the rate ofhydrolysis of the ester linkage increases with a decreasing value of m.

In the heterobifunctional polymer of the above formula, Q is a reactivemoiety capable of reacting with a reactive group in a target so as toform a linkage between the heterobifunctional polymer and the target. Atarget is defined below. Examples of Q include, but are not limited to,aldehydes, carboxylic acids, carbonate esters, hydrazides,N-succinimidyl esters, amines, isocyanates, alcohols, epoxide, thiols,orthopyridyl disulfides, and sulfonate esters.

Typically, Q reacts with a reactive group on a target to form a stablelinkage such that the heterobifunctional polymer of the invention isconjugated onto a target. A conjugate formed in this manner can berepresented by the formula:(CH₂═CZCO₂-POLY-W-L)_(x)-Tor(CH₂═CZCO₂-POLY-W-POLY′-L)_(x)-Twhere Z, POLY, POLY′ and W are as described above. L represents a stablelinkage formed as a result of the reaction between Q and a reactivegroup on T as described below. Examples of the hydrolytically stablelinkage L include, but are not limited to, amide from the reaction ofactive esters with amine, urethane from the reaction of isocyanate withalcohol, urea from the reaction of isocyanate with amine, amine from thereaction of aldehyde with amine and a reducing agent, amine from thereaction of epoxide with amine, and sulfonamide from the reaction ofsulfonate ester with amine.

T represents a target which is typically a molecule or an entity havinga desirable function or property. For example, T can be a protein or apharmaceutically effective agent. By forming a conjugate or hydrogel ofthe invention, a target T is in effect “loaded” onto a carrier and canbe delivered into a desired location under the protection of the polymerbackbone or the hydrogel matrix before it is released by hydrolyticbreakdown of the unstable linkage W in the polymer or hydrogel.

Accordingly, a target T in this invention can be any substance to whichit is desirable to link poly(alkylene oxide) or derivatives thereof. Tmust have a reactive group capable of reacting with the reactive moietyQ to form a stable linkage L. Examples of suitable Ts include, but arenot limited to, proteins, carbohydrates, lipids, hormones,oligonucleotides. Typically, T is a pharmaceutically effective agent.Examples of such substances include, but are not limited to, antibodiesand fragments thereof; cytokines including, but not limited tointerleukins (e.g., IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8,IL-9, IL-10, IL-11, and derivatives or fragments thereof), interferons(e.g., IFN-alpha, IFN-beta and IFN-gamma); growth factors, including butnot limited to colony stimulating factors, erythropoietins,haemopoietins, epidermal growth factors, platelet derived growthfactors, transforming growth factors, amphiregulin, somatomedin-C, bonegrowth factor, fibroblast growth factors, insulin-like growth factors,heparin binding growth factors, tumor growth factors and other growthfactors, platelet activating factors, macrophage activation factors, andother activating factors; transcription factors; substances affectingblood clotting including but not limited to heparin, proteases and theirpro-factors, clotting factors VII, VIII, IX, X, XI and XII, antithrombinIII, protein C, protein S, streptokinase, urokinase, prourokinase,tissue plasminogen activator, fibrinogen, hirudin, otherfibrinolytic/anticoagulant agents and other coagulation factors; lipidsincluding but not limited to phosphatidylethanolamine,phosphatidylserine, sphingosine, cholesterol and other steroids andderivatives thereof; nucleotides including but not limited toribonucleotides, deoxyribonucleotides, nucleosides, oligonucleotides,DNA, and RNA; enzymes; vaccines; vitamins; antibiotics; and otherpharmaceutically effective agents including but not limited toanthelminthic agents, noradrenalin, alpha adrenergic receptor ligands,dopamine receptor ligands, histamine receptor ligands,GABA/benzodiazepine receptor ligands, serotonin receptor ligands,leukotrienes and tri-iodothyronine and other small effector molecules,doxorubicin, methotrexate and other cytotoxic agents and derivativesthereof.

When the hydrolytically unstable linkage W is situated within thepoly(alkylene oxide) backbone of the heterobifunctional polymers or theconjugates of this invention, W can be formed by reacting two modifiedpolymers having terminal reactive moieties as illustrated below:-PEG-X+Y-PEG-→-PEG-W-PEG-In the above illustration, -W- represents the hydrolytically unstableweak linkage. X and Y represent the reactive moiety pairs as describedabove. Exemplary reactions are illustrated below where the reversereactions illustrate hydrolytic reversibility:

-PEG-CO₂H+HO-PEG-

-PEG-CO₂-PEG-(ester)+H₂O

-PEG-OPO₃H₂+HO-PEG-

-PEG-OPO₃(H)-PEG- (phosphate ester)+H₂O

-PEG-CHO+2(HO-PEG)-

-PEG-CH(O-PEG-)₂(acetal)+H₂O

-PEG-CHO+NH₂-PEG-

-PEG-CH═N-PEG-(imine)+H₂O

The hydrolytically stable linkage L can be formed, for example, throughthe following reaction:-PEG-W-Q+U-T→-PEG-W-L-T or-PEG-W-PEG-Q+U-T→-PEG-W-PEG-L-Twhere U is a reactive group on T.

The skilled artisan should recognize that when reference is made to an Xmoiety reacting with a Y moiety, or a Q group with a U group, additionalreagents or steps may be employed according to commonly acceptedchemical procedures and standards to achieve the desired linkage W or Las the case may be. There are many possible routes, too numerous tomention here, that could be taken and that should be readily apparent tothe skilled artisan. For example, one of skill in the art can beexpected to understand that when an alcohol and a carboxylic acid arereacted, the acid typically is converted to another form, the acidchloride, prior to reaction with alcohol. Several examples aredemonstrated in the Examples below.

The heterobifunctional polymers and conjugates of this inventiondescribed above can be employed in polymerization reactions to formpolymers and hydrogels.

Since the heterfunctional polymers and the conjugates of this inventionall have an acrylate group, vinyl polymerization of each of theheterfunctional polymers or conjugates can be conducted by a methodknown in the art. Two or more compounds selected from theheterfunctional polymers and the conjugates of this invention can becopolymerized. Many methods of vinyl polymerization are generally knownin the art and are useful in the present invention. Generally, when aconjugate is involved in the polymerization or copolymerization,conditions for the polymerization reaction should be selected such thatthe target in the conjugate is not adversely affected. Suitablepolymerization methods include, for example, redox initiation and photoinitiation. Other suitable methods should be apparent to a skilledartisan once apprised of the present disclosure.

In accordance with another aspect of this invention, hydrogels can beprepared from the heterobifunctional polymers and conjugates, as well asthe vinyl polymers by polymerization and/or crosslinking. As usedherein, “hydrogel” is intended to mean gels produced by incorporatingthe soluble hydrophilic polymers (e.g., heterfunctional polymers andconjugates of this invention) into a chemically crosslinked network ormatrix so that addition of water produces an insoluble swollen gel.Crosslinks can be formed from the heterobifunctional polymers orconjugates themselves. However, typically, crosslinks are introduced bycopolymerizing the heterobifunctional polymers or conjugates with amultiacrylate as a monomer. By “multiacrylate” it is intended to mean amolecule having two or more acrylate groups so that it can form acrosslinking bridge in vinyl polymerization of the heterobifunctionalpolymers or conjugates of the present invention. Preferably, themultiacrylate used is a PEG multiacrylate, i.e., a PEG molecule havingtwo or more acrylate groups therein. Exemplary examples of such PEGmultiacrylate can be, e.g.,CH₂═CHCO₂-PEG-O₂CCH═CH₂orCH₂═CHCO₂-PEG-O—CH₂CO₂CH(CH₃)CH₂CONH-PEGO₂CCH═CH₂.However, many other multiacrylate monomers can also be used as isapparent to a skilled artisan apprised of this invention.

Typically, a hydrolytically degradable conjugate of this inventionhaving a target therein is used in preparing the hydrogel of thisinvention. In this manner, the target is incorporated covalently intothe hydrogel which can be used as a carrier for in vivo delivery orother applications. Thus, the hydrogels of the invention areparticularly useful in drug delivery systems. By “drug” is meant anysubstance intended for the diagnosis, cure, mitigation, treatment, orprevention of disease in humans and other animals, or to otherwiseenhance physical or mental well being. For example, hydrogels made fromthe crosslinked PEG polymeric structures of the invention can be usedfor wound dressings. Wound dressings can be used internally to providedressings that degrade within the body over time.

In the hydrogel of this invention, the target material that iscovalently linked to the hydrogel can be released in an aqueousenvironment by hydrolytic breakdown of the hydrolytically unstablelinkage W. In order to control the rate of release of the target in thehydrogel, the unstable linkage W can be manipulated during thepreparation of the hydrogel. It has been discovered that the number ofatoms, particularly alkylene groups, proximate to the hydrolyzable bondin W affects the hydrolysis rate of the hydrolyzable bond. For example,as the number of methylene group increases, the hydrolysis ratedecreases.

For example, when W has the structure of:—O(CH₂)_(m)—CO₂R₁—CO₂—where m ranges from 1 to 10, R₁ is selected from the group consisting of—CH₂—, —CH₂CH₂—, and —CH(CH₃)CH₂—, increasing the m value decreases thehydrolysis rate of esters and increases the time required for the gel todegrade. Typically, if m in the above example is 1, then the esterlinkages of the gel will hydrolyze with a half life of about 4 days atpH 7 and 37° C. If m is 2, then the half life of hydrolytic degradationof the ester linkages is about 43 days at pH 7 and 37° C. Phosphateesters, acetals, imines, and other hydrolytically unstable linkages canbe similarly formed and the hydrolysis rate can be similarly controlledby controlling the number of methylene groups adjacent thehydrolytically unstable linkage.

In addition, in the hydrogel of the present invention, the release rateof the target from the hydrogel can also be controlled by varying thenumber x of the PEG acrylates linked to the target. The release rate ofthe target from the hydrogel is decreased when the number of PEGacrylates linked to the target is increased. Release rate is increasedby decreasing the number.

In the hydrogel of this invention, a two-fold control of the targetrelease rate is made possible: (1) by varying the number of atomsproximate to the hydrolyzable bond in the hydrolytically unstablelinkage W; and (2) by controlling the number of the PEG acrylates linkedto the target. As a result, the hydrogels of this invention can bedesigned to have a more precisely controlled target release rate, whichis useful in hydrogel applications, e.g., drug delivery.

The following examples are given to illustrate the invention, but shouldnot be considered in limitation of the invention.

Example 1. Synthesis of CH₂═CHCO₂-PEG-OCH₂CO₂CH(CH₃)CH₂CO₂NS

-   -   (NS═N-succinimidyl)        Example 2. Modification of proteins        Example 3. Preparation of gels        Example 4. Release of proteins from gels

EXAMPLES Example 1 Preparation of CH₂═CHCO₂-PEG-OCH₂CO₂CH(CH₃)CH₂CO₂NS

Reaction Scheme:BzO-PEG-OCH₂CO₂H+SOCl₂→BzO-PEG-OCH₂COCl+SO₂+HCl(Bz=Benzyl)BzO-PEG-OCH₂COCl+HOCH(CH₃)CH₂CO₂H→→BzO-PEG-OCH₂CO₂CH(CH₃)CH₂CO₂H+HClBzO-PEG-OCH₂CO₂CH(CH₃)CH₂CO₂H+H₂→→HO-PEG-OCH₂CO₂CH(CH₃)CH₂CO₂H+BzHHO-PEG-OCH₂CO₂CH(CH₃)CH₂CO₂H+CH₂═CHCOCl+2(CH₃CH₂)₃N→→CH₂═CHCO₂-PEG-OCH₂CO₂CH(CH₃)CH₂CO₂ ⁻+2(CH₃CH₂)₃NH′+Cl⁻CH₂═CHCO₂-PEG-OCH₂CO₂CH(CH₃)CH₂CO₂ ⁻(CH₃CH₂)₃NH⁺+a.) Preparation of BzO-PEG-OCH₂CO₂—CH(CH₃)CH₂CO₂H

BzO-PEG-OCH₂CO₂ H (MW=3400, 15 g, 4.4 mmole) was azeotropically driedwith 60 ml of toluene under N₂. After two hours, the solution was slowlycooled to room temperature. To this solution was added thionyl chloride(18 ml, 36 mmole). The resulting solution was stirred overnight, thesolvent condensed by rotary evaporation, and the syrup dried in vacuofor about four hours over P₂O₅ powder. 3-hydroxybutyric acid (1.45 g,13.5 mmole) was azeotropically dried with 70 ml of 1,4-dioxane, and thenadded to the dried BzO-PEG-OCH₂COCl. After the PEG acyl chloride haddissolved, 4.5 ml of dry triethylamine was injected into the system andthe solution was stirred overnight. The salt was removed by filtrationand the filtrate was condensed on a rotary evaporator at 55° C. anddried in vacuo. The crude product was then dissolved in 100 ml ofdistilled water and the pH of the solution was adjusted to 3.0. Theaqueous phase was extracted three times with a total of 80 ml ofmethylene chloride. The organic phase was dried over sodium sulfate,filtered, condensed on a rotary evaporator, and precipitated into 100 mlof ethyl ether. The product was collected by filtration and dried invacuo at room temperature. Yield 14 g (93%). ¹H nmr (DMSO-d₆): δ 3.5 (brm, PEG), 2.58 (d, -PEGCOOCH(CH₃)CH₂COOH), 5.14 (h,-PEG-COOCH(CH₃)CH₂COOH), 1.21 (d, —PEGCOOCH(CH ₃)CH₂COOH), 4.055 (s,PEGOCH ₂COO), 4.49 (s, _(C)6H₅—CH ₂—OPEG-), 7.33 (s+comp. mult., C₆ H₅—CH₂—OPEG-).

b.) Preparation of HO-PEG-OCH₂CO₂—CH(CH₃)CH₂CO₂H

A solution of BzO-PEG-OCH₂CO₂-PEG-OCH(CH₃)CH₂CO₂H (8 g) in benzene (50ml) was hydrogenolyzed with H₂ (2 atm) on 4 gram Pd/C (10%) at roomtemperature for 48 hours. The catalyst was removed by filtration, thesolvent was condensed, and the solution was precipitated into ethylether. The product was collected by filtration and dried in vacuo atroom temperature.

Yield: 6.6 gram (83%). ¹H nmr (DMSO-d₆): δ 3.5 (br m, PEG), 2.51 (d,—PEGCO₂CH(CH₃)CH ₂CO₂H), 5.16 (h, -PEG-CO₂CH(CH₃)CH₂CO₂H), 1.22 (d,-PEG-CO₂CH(CH₃)CH₂CO₂H), 4.06 (s, -PEGOCH ₂CO₂PEG-).

c.) Preparation of CH₂═CHCO₂-PEG-OCH₂CO₂—CH(CH₃)CH₂CO₂H

HO-PEG-OCH₂CO₂CH(CH₃)CH₂CO₂H (3 g, 0.88 mmole) was azeotropicallydistilled with 40 ml of toluene under N₂ until approximately 15 ml ofsolution remained. The solution was then cooled to room temperatureunder N₂ and 25 ml of methylene chloride and triethylamine (1.5 mmole)were added. The solution was cooled in an ice bath and acryloyl chloride(2 mmole) were added dropwise. After addition of acryloyl chloride, theice bath was removed and the solution was stirred at room temperatureovernight. The methylene chloride was then partially removed undervacuum, the salt was removed by filtration, and the filtrate added to100 ml of ether. The precipitated product was collected by filtrationand dried in vacuo. The product was then dissolved in sodium acetatebuffer (0.1M, pH 5.5), stirred for half an hour, and extracted threetimes with methylene chloride. The organic phase was dried over sodiumsulfate, filtered, condensed, and precipitated in 100 ml of ethyl ether.The precipitate was collected by filtration and dried in vacuo at roomtemperature. Yield 2.4 g (80%). ¹H nmr (DMSO-d₆): δ 3.5 (br m, PEG),2.51 (d, CH ₂CO₂H), 5.16 (h, —CH(CH₃—), 1.22 (d, —CH(CH ₃)—), 4.06 (s,PEGOCH₂CO₂PEG-), 4.21 (t, —CO₂CH ₂CH₂O—), 5.85-6.45 (m, CH ₂═CH—).

d.) Preparation of CH₂═CHCO₂-PEG-OCH₂CO₂—CH(CH₃)CH₂CO₂NS

CH₂═CH—CO₂-PEG-OCH₂CO₂CH(CH₃)CH₂CO₂H (1.4 g, approx. 0.4 mmole) andN-hydroxysuccinimide (51 mg, 0.43 mmole) was dissolved in 30 ml of drymethylene chloride. To this solution was added dicyclohexylcarbodiimide(95 mg, 0.45 mmole) in 5 ml of dry methylene chloride. The solution wasstirred under nitrogen overnight and the solvent removed by rotaryevaporation. The resulting syrup was dissolved in 10 ml of dry tolueneand the insoluble solid was removed by filtration. The filtrate wasadded to 100 ml of dry ethyl ether and the precipitated product wascollected by filtration and dried in vacuo at room temperature.

Yield 0.94 g (94%). ¹H nmr (DMSO-d₆): δ 3.5 (br m, PEG), 3.0-3.2 (m,-PEGCOOCH(CH₃)CH ₂COONS), 5.26 (h, PEGCOOCH(CH₃)CH₂COONS), 1.3 (d,-PEGCOOCH(CH ₃)CH₂COONS), 4.10 (s, -PEGOCH ₂COO(CM)), 2.81 (s, NS), 4.21(t, CH₂═CH—COO—CH ₂CH₂—O-PEG-, 4H), 5.85-6.45 (m, CH ₂═CHCOOPEG-).

Example 2 Modification of Proteins

a) Modification of Lucifer-Yellow Modified Lysozyme

CH₂═CHCO₂-PEG-OCH₂CO₂—CH(CH₃)CH₂CO₂NS (19 mg, 5.5 mmole) was dissolvedin 0.1 ml of water and 0.5 ml of lucifer-yellow modified lysozymesolution and (20 mg/ml) in borate buffer (0.1M, pH 8.0) was added. Thesolution was shaken gently on an auto-shaker at room temperature for 3hours. Completion of the reaction was demonstrated by capillaryelectrophoresis. The solution was then stored at 4° C. prior to releasestudies.

b) Modification of Fluorescein Isothiocyanate-Bovine Serum Albumin(FTIC-BSA):

CH₂═CHCO₂-PEG-OCH₂CO₂—CH(CH₃)CH₂CO₂NS (9.3 mg, 2.7 mmole) was dissolvedin 0.5 ml of deionized water and 1.5 ml of FITC-BSA solution (15 mg/ml)in boric buffer (0.1M, pH 8.0) was added. The solution was shaken gentlyon an auto-shaker at room temperature for 3 hours. Completion of thereaction was demonstrated by capillary electrophoresis. The solution wasthen stored at 4° C. prior to release studies.

Example 3 Preparation of Gels

a.) By Redox Initiation

A solution of (0.5 ml, 200 mg/ml in water ofCH₂═CHCO₂-PEG-O—CH₂CO₂CH(CH₃)CH₂CONH-PEGO₂CCH═CH₂ orCH₂═CHCO₂-PEG-O—CH₂CO₂PEG-O₂CCH═CH₂, and 0.5 ml of buffered PEGacrylate-modified lucifer yellow lysozyme (Example 2a) solution (10mg/ml, f and 20 ml of potassium persulfate (K₂S₂O₈, 100 mM) were mixed.To the solution was added 20 ml of iron sulfate (FeSO₄, 100 mM). Afterrapid shaking, a gel formed in a few minutes.

A suitable buffer for this procedure is boric buffer (0.1 M) orphosphate buffer (<0.01M) with pH range of 6 to 8.

b.) By Photo Initiation

Difunctional PEG acrylate solution (0.5 ml, 400 mg/ml in water,CH₂═CHCO₂-PEG-O CH₂CO₂CH(CH₃)CH₂CONH-PEGO₂CCH═CH₂ orCH₂═CHCO₂-PEG-O—CH₂CO₂PEG

—O₂CCH═CH₂, 0.5 ml of buffered (pH 7) PEG acrylate-modified FTIC-BSAsolution (Example 2b) and 100 ml of 2,2-dimethoxy-2-phenyl-acetophonesolution (10 mg/ml in ethanol) were mixed. The solution was exposed toUV radiation at a wavelength of 360 nm and the gel formed in about 10minutes.

Example 4 Release of Proteins from the Gels

The release of lucifer yellow lysozyme was monitored using a flow UVspectrophotometer at 428 nm and 37° C. in 0.1 M phosphate buffer (pH 7).Release profiles for two experiments are shown in FIG. 1.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

That which is claimed is:
 1. A hydrogel comprising, a co-polymerizationproduct of a PEG multiacrylate and a heterobifunctional PEG conjugatewherein, (i) the multiacrylate has the formula ofCH₂═CHCO₂-PEG-O₂CCH═CH₂, wherein PEG is polyethylene glycol, and (ii)the conjugate has the formula of (CH₂═CZ—CO₂-PEG-W-L)_(x)-T, wherein Zis an alkyl group or hydrogen atom, W is a hydrolytically unstablelinkage comprising a hydrolysable covalent bond, L is a covalentlinkage, PEG is a polyethylene glycol, T is a protein, and x is numberfrom 1 to 10.